1. Field of the Invention
This invention provides novel attenuated strains of pathogenic mycobacteria and, in particular, of Mycobacterium tuberculosis and M. bovis. Attenuation is achieved by downregulating or eliminating the expression of the xcex1-crystallin heat shock protein gene (xe2x80x9cacr genexe2x80x9d). The invention also provides vaccines and methods of vaccinating mammals for protection against mycobacterial diseases including, in particular, tuberculosis.
2. Relevant Art
Pathogenic mycobacteria are the causative agents of a number of human and animal diseases. For example, tuberculosis is a health problem of considerable importance in the human population. Recent estimates are that as much as one-third of the population of the world is infected with M. tuberculosis, that there are 30 million active cases, that there are some 10 million new cases annually, and that tuberculosis causes some 6 percent of all deaths worldwide. See, e.g., Daniel, T., Tuberculosis in Harrison et al., eds., Principles of Internal Medicine, McGraw-Hill, Inc., New York, N.Y. (13th ed., 1994) (hereafter xe2x80x9cHarrison""s (1994)xe2x80x9d; the entirety of Harrison""s is hereby incorporated by reference). At present, the only vaccine is an attenuated strain of M. bovis designated bacillus Calmette-Guerin (xe2x80x9cBCGxe2x80x9d). While this vaccine is considered safe, its efficacy is still uncertain. Ibid.
BCG is created by multiple passages of the M. bovis organism. These multiple passages are considered necessary to attenuate the pathogenicity of the organism but, as the organism adapts to laboratory passaging, it undergoes undefined and unknown changes from that of the wild type. These changes, in turn, diminish the relevance of the immune response generated by use of BCG to subsequent challenge by wild type, pathogenic bacteria, and are probably due at least in part to the continuing uncertainty over its efficacy. Moreover, since M. bovis infects cows, rather than humans, a portion of the immune response it raises is not directed against antigens relevant to human disease.
What is therefore needed is a method of producing attenuated mycobacteria without the multitude of passages currently necessary to produce BCG, so that the immune response generated is more relevant to the challenge posed by wild type, pathogenic bacteria. What is also needed, in particular, is a vaccine for tuberculosis based on mycobacteria less removed from the wild type than are those used to create BCG. What is even more needed is a vaccine which is based on M. tuberculosis, and which can therefore be expected to raise an immune response more directly relevant to the antigens presented by wild type M. tuberculosis. It would further be desirable to have a vaccine of organisms capable of replicating for a short time in a vaccinated host, to permit the organism to present proteins and other antigens which may only be presented live, metabolizing organisms, but also capable of being completely contained by the host. The present invention provides these and other advantages.
This invention provides a vaccine for protection against tuberculosis. The vaccine comprises pharmaceutically acceptable excipients and Mycobacterium sp. attenuated by having the expression level of the xcex1-crystallin heat shock protein gene reduced by at least 75% of the wild type expression level, said Mycobacterium sp. being present in a concentration effective to provide immunoprotection to a host mammal. In preferred embodiments, expression of the xcex1-crystallin heat shock protein gene is eliminated. In more preferred forms, the gene for the protein is xe2x80x9cknocked out.xe2x80x9d
In one embodiment, the vaccines have the Mycobacterium sp. in an isotonic salt solution. Preferred dosages are between 1 and around 1,000,000 cells per dose. More preferred dosages are between around 100 and around 100,000 cells per dose. Somewhat more preferred are between around 100 and around 50,000 cells per dose. Even more preferred are between around 100 and around 25,000 cells per dose. Most preferred are between around 100 and around 1,000 cells per dose.
This invention also provides for a method of vaccinating a mammal susceptible to an infection of Mycobacterium sp. The method comprises the administration of an amount of Mycobacterium sp. attenuated by having the expression level of xcex1-crystallin heat shock protein gene reduced by at least 75% of the wild type expression level, said administration of Mycobacterium sp. in an amount effective to provide immunoprotection to a host mammal. Preferred routes of administration are via the nasal passages or via parenteral injection. The preferred dose is between 100 and 100,000 cells, depending, inter alia, on the route of administration.
In addition to vaccines and methods of vaccinating, this invention provides for novel strains of Mycobacterium sp. wherein the expression level of the xcex1-crystallin heat shock protein gene is reduced by at least 75% compared to the wild type expression level. In one embodiment, the strains are housed in a sealed vial where the vial is packaged with instructional material stating a method of injecting the strain into a human to provide the human with immunoprotection from tuberculosis.
In one embodiment of the vaccine, methods, and strains described above, the Mycobacterium species is selected from the group consisting of M. tuberculosis and M. bovis. 
Additionally, the invention provides a method of manufacturing attenuated mycobacteria by reducing the expression of the xcex1-crystallin heat shock protein gene by at least 75% compared to the wild type expression level. In one embodiment, the reduction is by deletion of the gene encoding the protein. In preferred embodiments, the mycobacteria manufactured by this method are M. tuberculosis or M. bovis. 
Further, the invention provides a method for attenuating virulence of a Mycobacterium sp., wherein the method comprises reducing the expression of the xcex1-crystallin heat shock protein gene by at least 75% compared to the wild type expression level. In one embodiment, the reduction is by deletion of the gene encoding the protein. In preferred embodiments, the mycobacteria whose virulence are attenuated by this method are M. tuberculosis or M. bovis. 
xe2x80x9cAttenuatedxe2x80x9d refers to a state of reduced pathogenicity of an organism that in its wild state is pathogenic. It is usually measured by an inhibition, partial or complete, of the ability of a pathogenic organism to colonize, infect, grow, reproduce, or survive in a host. When attenuation is due to a defined genetic change, that change may be a gene deletion, replacement, or alteration, transcriptional inhibition, or translational inhibition.
xe2x80x9cImnmunoprotectionxe2x80x9d refers to the ability of the immune system of a mammal such as a human to reduce infection of a pathogen such as M. tuberculosis or M. bovis when challenged with what would otherwise be an infectious dose.
xe2x80x9cIsotonic salt solutionsxe2x80x9d are salt solutions that are osmotically balanced against the cells in which they are contacted. They are preferably at physiologic pHs of between 6.5 and 7.5.
xe2x80x9cInstructional materialxe2x80x9d refer to written, audio or visual means to convey the proper use of material or pharmaceuticals, vaccines or reagents.
xe2x80x9cPharmaceutically acceptablexe2x80x9d means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the acr attenuated mycobacteria without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition.
The phrase xe2x80x9cexpression level of xcex1-crystallin heat shock protein gene reduced by at least 75% of the wild type expression levelxe2x80x9d refers to a reduction of at least 75% in the relative level of expression of xcex1-crystallin heat shock protein between a wild type pathogenic strain and a strain deliberately altered to reduce natural levels of the protein, measured by any scientifically valid technique. Typically, measurements of relative level involve labeling antibodies specific to the xcex1-crystallin heat shock protein or labeling probes which specifically hybridize to nucleic acids encoding the protein in genomic DNA or mRNA. Methods of altering the relative level of expression are not limited to gene xe2x80x9cknock outxe2x80x9d and include methods of interference with mRNA transcription or with translation, including interference with translation by means of antisense or ribozyme technology.
As used herein, xe2x80x9cstrainxe2x80x9d has two meanings. With respect to a wild type microorganism, xe2x80x9cstrainxe2x80x9d refers to a substantially pure culture of a mycobacterial species, and is commonly applied to a culture of mycobacteria isolated from a patient or cluster of patients manifesting disease. With respect to a microorganism whose expression of the xcex1-crystallin heat shock protein has been altered from that of the corresponding wild type, such as recombinantly altered organisms, xe2x80x9cstrainxe2x80x9d refers to a substantially pure culture of a mycobacterial species wherein the microorganisms are at least 70% homogeneous in their expression of the xcex1-crystallin heat shock protein. More preferably, at least 80% of the microorganisms are homogeneous in their expression of this protein. Even more preferably, the homogeneity is around 90% or above. Most preferably, the microorganisms are homogeneous in not expressing detectable levels of this protein.
xe2x80x9cVialxe2x80x9d refers to any container that is sealed from the atmosphere and able to hold within it a liquid or freeze dried material without measurable losses.
This invention arises from the surprising discovery that Mycobacterium sp. such as M. tuberculosis and M. bovis can be attenuated by downregulating the expression of the xcex1-crystallin heat shock protein gene. More surprisingly this novel attenuated strain has the ability to confer immunoprotection to animals susceptible to infections from Mycobacterium sp.
1. Obtaining Forms of Mycobacterium sp. Attenuated by Downregulating Or Eliminating Expression of Acr
To obtain attenuated forms of Mycobacterium sp., one starts with standard pathogenic strains of organisms such as M. tuberculosis or M. bovis. Strain H37Rv is typical of the pathogenic forms of M. tuberculosis. Qualified individuals can obtain this strain from the American Type Culture Collection, located at 10801 University Boulevard, Manassas, Va., USA. The accession number is 27294. Other strains are available from Colorado State University, Fort Collins under the NIAID xe2x80x9cTuberculosis Research Materialsxe2x80x9d contract (administered by Dr. Ann Ginsberg, Tuberculosis Program Officer, NIAID, Solar Building, Room 3A31, Bethesda Md. 20892.
To maximize immunoprotection from a vaccination, it is generally desirable to maximize exposure to antigens present in virulent strains of the wild-type organism. Therefore, while various species and strains of mycobacteria can be used, it is preferable to use M. tuberculosis or M. bovis. M. tuberculosis is the most preferred species where the attenuated form is intended to vaccinate humans against tuberculosis, since it is the form most relevant to the human disease. It is further preferable to use a strain which has been isolated relatively recently from clinical specimens (that is, a strain which has not undergone a large number of passages). Such a strain is CSU93, a recently isolated hypervirulent strain. CSU93 is available to qualified investigators from Dr. John Belisle of Colorado State University under the terms of the NIAID contract mentioned above. Strains of mycobacterium already attenuated in other aspects can also be used. M. bovis bacillus Calmette-Guerin (xe2x80x9cBCGxe2x80x9d), which is considered to have multiple, but unknown, alterations due to multiple passaging, can be used and, indeed, can be expected to require less passaging before being suitable for use because of the reduced pathogenicity imparted by the reduction or elimination of the xcex1-crystallin heat shock protein. BCG would, however, be expected to produce a less robust immune response than a more recent isolate and is therefore less preferred.
The target gene, xcex1-crystallin heat shock protein gene (xe2x80x9cacr genexe2x80x9d) is known and its sequence is a matter of public knowledge. The acr gene was first published in 1992 by two groups (Verbon et al., J. Bacteriol. 174: 1352-1359 (1992); Lee et al., Infect. Immun. 60: 2066-2074 (1992)). The 14,000-molecular weight antigen of M. tuberculosis is related to the xcex1-crystallin family of low-molecular weight heat shock proteins. The amino acid and nucleic acid sequences are published in GenBank and have the following GenBank Accession Numbers; S79751 and M76712.
Mycobacterium sp. can be maintained under appropriate culture conditions (see, e.g., Yuan et al., J. Bacteriol. 178: 4484-4492 (1995)). While the organisms can be grown on any of a large number of media well known to those of skill in the art, a preferred medium is Middlebrook 7H9 (Remel Laboratories, Lenexai, Kans.), supplemented with albumin-dextrose complex (xe2x80x9cADCxe2x80x9d), available from Difco (Detroit, Mich.).
There are a number of conventional methods for selectively downregulating the expression of a specific protein. These include gene removal, replacement, transposition, antisense technology, and ribozyme technology.
Gene deletion is a preferred method because the gene is fully disabled by removal. The acr gene is contained entirely within a 4 kb BamHI restriction fragment. One way to eliminate production of the protein is to replace the gene encoding this protein with an antibiotic resistance cassette such as hygromycin or kanamycin. These resistance cassettes have been previously described in George et al., J. Biol. Chem. 270: 27292-27298 (1995). The antibiotic marker can be used to replace a segment of the gene or the whole gene. In this case, the acr gene is contained entirely on a 1 kb EcoRV fragment that is internal to the 4 kb BamHI fragment described above. Restriction digestion of the BamHI fragment with EcoRV removes the 1 kb EcoRV fragment. The resulting ligation of a blunt-ended antibiotic resistance cassette in place of the 1 kb EcoRV fragment into the BamHI fragment produces a construct lacking the xcex1-crystallin gene. Transformation of Mycobacterium sp. with either linear or supercoiled DNA will result in gene replacement in a small fraction of the resulting organisms. Those organisms can then be selected by testing for antibiotic resistance added by the replacement cassette and cultured to provide the quantities of attenuated organisms desired.
Antisense technology is another viable method for downregulation of acr gene expression. Expression of the xcex1-crystallin gene can be interrupted at the level of transcription by overproducing a small segment of RNA encoding a sequence contained on the sense strand of the normal xcex1-crystallin gene. Overproduction of, for example, the first 30 nucleotides of the gene would result in an inhibition of transcription of the normal acr gene.
Gene knockout of the acr gene can also be achieved via transposition. Insertional mutagenesis of the acr gene could be achieved using a number of different random transposition systems. Such a system might be the recently described IS1096-derived transposon by McAdam et al., Infection and Immunity 63: 1004-1012 (1995). Such transposons would be marked with a suitable resistance gene, such as kanamycin, hygromycin, streptomycin or others, and random transposants would be selected for antibiotic resistance before screening for transposition within the acr gene.
Ribozyme technology is a ready means to selectively target specific mRNA for cleavage prior to its being expressed. A general review of this technology is found in Castanotto et al., Crit. Rev. Eukaryotic Gene Expression 2: 331-357 (1992), Castanotto et al., Advances in Pharmacology 25:289-317 (1994), and Haseloff and Gelach, Nature 334:585-591 (1988). In general there are two main types of available ribozymes, hammerhead and hairpin ribozymes. See, e.g., Rossie et al., Pharmac. Ther. 50:245-254 (1991) (hammerhead ribozymes) and Hampel et al., Nucl. Acids Res. 18:299-304 (1990); U.S. Pat. No. 5,254,678 (hairpin ribozymes). Either form can be modified in its substrate binding regions to selectively bind to and cleave mRNA encoding the acr gene. At least 30 ribozymes have been demonstrated to be effective in cells at disabling a variety of viral and endogenous substrates, as tabulated by Stull and Szoka (Pharma. Res. 12(4):465-483 (1995)). More specific information on creating optimized ribozymes for a select target, such as the xcex1-crystallin heat shock protein, can be found in WO 96/01314, U.S. Pat. No. 5,496,698, and Campbell and Cech, RNA 1: 598-609 (1995). In brief, genes encoding ribozymes are transfected into the Mycobacterium sp. and are expressed at a level that directs the desired reduction of expression.
2. Determining Attenuation of the Acr Gene
Once a Mycobacterium sp., such as M. tuberculosis or M. bovis, has been modified to reduce the expression levels of acr gene, it is desirable to monitor and measure the level of reduction. Our studies have demonstrated the ability to completely eliminate the expression of the gene. The reduction, however, does not need to be 100% complete to render the attenuated strain useable as a vaccine. The reduction need only be sufficient to reduce pathogenicity to a level that eliminates clinical manifestation of disease, while still evoking a protective immune response.
There are several standard means to measure and quantify expression levels of the acr gene. These include Southern, Northern, and Western blotting techniques. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes part I, chapter 2 (1993). Other suitable references for detection of nucleic acids are Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. 1989) and Current Protocols in Molecular Biology, (Ausubel et al., eds., 1987) .
For a Southern blot analysis, one simply selects a region of the xcex1-crystallin heat shock protein gene as a probe (about 500 bases or more in length) and analyzes the chromosomal DNA from a test sample of potentially attenuated Mycobacterium sp. The DNA is digested with any number of different restriction enzymes and then transferred to a filter such as nitrocellulose. The transferred DNA is then screened by hybridization with the above probe, which has been radiolabelled using standard, commercially prepared reagents. An attenuated strain would be characterized by a lack of, or reduced levels of, hybridization with the xcex1-crystallin heat shock protein gene specific probe and/or a corresponding change in restriction pattern as detected by the probe and/or a corresponding change in the size of the restriction fragments as compared to wild type. In addition, a probe that corresponds to the inserted nucleic acid can be used as a positive marker for gene replacement.
Alternatively, one could use a Western blot analysis to determine reduced levels of xcex1-crystallin heat shock protein. The xcex1-crystallin heat shock protein is a major cell surface protein of M. tuberculosis and has been isolated and purified (see, e.g., Yuan et al., J. Bacteriol. 178: 4484-4492 (1996)) (hereafter, Yuan et al., 1996). Specific polyclonal and monoclonal antibodies are prepared using conventional techniques. The antibodies are then used to screened against cell lysates of attenuated Mycobacterium sp. which have been disrupted and separated using any of a variety of electrophoretic separation techniques. Antibodies to xcex1-crystallin heat shock protein are available. The monoclonal designated F22-2 has been deposited with the Centers for Disease Control/World Health Organization Collection of Antibodies to mycobacteria.
One could also use Northern blot analysis to measure changes in transcription levels of acr gene. The total RNA is isolated from cells using standard techniques, typically by using strong disruptants. The RNA is separated according to size using any of a number of conventional electrophoresis techniques and transferred to a filter; the RNA is then probed with a labeled probe specific for the coding region of the acr gene. For example, one can obtain a suitable probe by using polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) technology to amplify a subsequence of the coding sequence of the GenBank-listed nucleotide sequence provided above. The following primer pair is suitable for this purpose: nucleotides 11-30 and 451-470. An attenuated strain would be characterized by a lack of, or reduced level of, hybridization with the probe, detection of a transcript with an altered size as compared to wild type, or both. In addition, a probe that corresponds to the inserted nucleic acid can be used as a positive marker for gene replacement.
Yet another alternative is to use amplification of the DNA or RNA, by PCR, ligase chain reaction (xe2x80x9cLCRxe2x80x9d), or the like. Standard techniques for performing such assays are known in the art (see, e.g., Eckert and Kunkel, PCR Methods and Applications 1: 17 (1991); Innis, M., et al., eds., PCR Protocols 1990 (Academic Press, San Diego Calif.);Wallace et al., Ligase Chain Reaction, in Pfiefer, G., ed., Technologies for Detection of DNA Damage and Mutations (Plenum Publishing Corp, New York, N.Y., 1996), pp. 307-322). When RNA is amplified it is first reverse transcribed into cDNA. The DNA (either cDNA or genomic DNA) is then amplified under controlled conditions using a suitable pair of primers to yield reproducible and quantifiable data for determining the original amount of xcex1-crystallin heat shock protein gene specific transcript or gene present in a test sample. As described above, an attenuated strain would be characterized by amplification bands that have altered sizes or that are missing. In addition, the replacement nucleic acid can also be amplified as a positive confirmation that the acr gene has been replaced.
3. Formulation of Vaccines
Having downregulated the expression of xcex1-crystallin heat shock protein in a Mycobacterium sp. strain and having evidence that the downregulation is sufficient to produce an attenuated strain of Mycobacterium sp., one then formulates the vaccine.
The attenuated strains can be raised in, for example, Middlebrook 7H9 liquid media supplemented with albumin-dextrose complex, 0.2% glycerol, and 0.05% Tween-80, as described in Yuan et al., (1996). Other media known in the art can, however, be used. Cultures are typically begun by inoculation from single colonies of organisms confirmed to be acr gene disruptants. If the organisms have had the acr gene replaced with a gene conferring resistance to an antibiotic, then the organisms will typically be grown on media supplemented with the antibiotic to which resistance-has been conferred, for example, 50 xcexcg/ml hygromycin when the target gene has been replaced by a hygromycin resistance gene. The cultures are grown to an optical density (read at 650 nm) of 1.0, which corresponds to a cell density of about 1xc3x97109 colony forming units per ml of Mycobacterium sp.
For vaccine administration, the acr attenuated mycobacteria are typically combined with a pharmaceutically acceptable carrier appropriate for the intended mode of administration. Pharmaceutical compositions include a unit dose of the acr attenuated mycobacteria in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. The proportion of acr attenuated mycobacteria and adjuvant can be varied over a broad range so long as both are present in effective concentrations as described below.
Examples of pharmaceutically acceptable carriers include solutions such as normal saline, Ringer""s solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts, with or without sugar additives such as glucose. Suitable carrier solutions also include, for example, water, dextrose, glycerol, or buffered vehicles. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Adjuvants are generally commercially available from various sources.
4. Administration of the Vaccines
The pharmaceutical compositions of the invention can be administered by any suitable means, e.g., parenterally (subcutaneously, intramuscularly, or intraperitoneally), intravenously, nasally or orally. Preferably, the compositions are administered nasally, with aerosol administration being a preferred mode. Injectables can be prepared in conventional forms. An appropriate evaluation of the time and method for delivery of the vaccine is well within the skill of the clinician.
Vaccine compositions of the invention are administered to humans and other mammals susceptible to or otherwise at risk of infection to elicit an immune response against the mycobacteria and thus enhance the patient""s own immune response capabilities. Such an amount is defined to be an immunogenically effective amount.
The exact amount of such pharmaceutical compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular mycobacteria used, the mode of administering it, the nature of the formulation, and the like. The judgment of the prescribing physician is also an important consideration. Generally, dosage will approximate that which is typical for the administration of other live attenuated vaccines (see, e.g., Ravn et al., J. Immunol. 158: 1949-1955 (1997) (administration to humans); Chambers et al., J. Immunol. 158: 1742-1780 (1997) (administration to mice); Roia, F. Immunizing Agents and Diagnostic Skin Antigens, in Gennaro, A., ed., Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. (17th ed., 1985 and 18th ed., 1990) (the entirety of both of these editions of Remington""s are hereby incorporated by reference).
Natural infections are believed to occur from exposure to as few as 1 to 10 organisms. Clearly, however, the chances of becoming infected go up the number of organisms increases, although there can be expected to be a point of diminishing returns. For purposes of vaccination, the number of acr attenuated mycobacteria for an aerosol administration can range from 1 to as many as about 106 cells per dose. Preferably, the number of cells administered will range between about 102 and about 104 cells per dose, and more preferably are between about 102 and about 103 cells per dose. For parenteral administration, the number of cells can also range from about 1 to about 106 cells per dose, with about 104 cells per dose being preferred. For oral administration, the dose range is preferably about 104 to about 107 cells, with doses on the lower end of the range being preferred. A suitable dose size is about 0.1-5 ml, preferably about 0.1-1 ml. Accordingly, a typical dose for aerosol administration for example, would comprise 0.1 to 0.5 ml containing about 1xc3x97103 cells, or for subcutaneous administration, 0.1 to 0.5 ml containing about 1xc3x97104 cells.
The timing of administration of the vaccine and the number of doses required for immunization can be determined from standard live attenuated vaccine administration protocols. Typically, this vaccine composition will be administered in one dose (see, e.g., Product Information, Physicians Desk Reference (1996); Remington""s, supra).
Lyophilization of the bacterial cells, and resuspension in a sterile solution prior to administration is will be a preferred embodiment of the invention in countries in which availability of refrigeration in the field or rural areas may be problematic, and is likely to be preferred in this country where ease of storage is desired. Frozen aliquots of vaccine, which can be thawed and injected without the need for resuspension, are likely to be preferred where these two factors are not major concerns since freezing results in better viability of the organisms than does lyophilization.
5. Confirming Immunization
Confirmation of immunization using the vaccine of the invention is typically performed by the tuberculin test. The standard antigen for the test is tuberculin purified protein derivative (xe2x80x9cPPDxe2x80x9d), also known as xe2x80x9cold tuberculin.xe2x80x9d This antigen is a heterogenous and undefined substance prepared from mycobacteria culture filtrates. PPD is prepared according to standard procedures (see, e.g., Landi, Applied Microbiol. 11: 408 (1963)). More purified antigens are also available and may be used in place of PPD.
A standardized dose of PPD antigen is typically administered via an intracutaneous injection in the forearm in what is often called the tuberculin, or Mantoux, test (see Harrison""s (1994) at 714-715; McMurray, D., in Baron, ed., Medical Microbiology, Churchill Livingstone, New York, N.Y. (3d ed., 1991) at 459). Multiple intracutaneous puncture or tine tests are also used.
The immune reaction to the antigen is read by measuring the transverse diameter of induration (the raised, hard area of inoculation with the antigen) at 48 to 72 hours. As a test of immunization (not diagnosis), an induration size of 5-10 mm is generally considered doubtful and an induration size of 10-15 mm is considered a successful immunization. Typically, the size of an induration on an immunized person will be greater than 15 mm. Because PPD reactivity seems to decrease with age, it may be useful to administer a repeat test on an older person with an intermediate dose of PPD 7 to 10 days after the first test. According to Harrison""s (1994), a reaction to the second test should be considered significant. False negative tests are common, often resulting from technical errors such as subcutaneous injection, use of outdated PPD, or permitting PPD to remain in the syringe before use.
6. Alternative Uses for Attenuated Strains
Production of human vaccines against tuberculosis is an important part of this invention; however that is not the only use of the attenuated strains of the invention. Attenuated forms of M. bovis (which, as noted in the Background section, supra, infects cows), can also be used, for example, as a veterinary vaccine to protect cows and other animals against that organism. Other pathogenic mycobacteria expressing the acr protein and acr homologs, such as the members of the tuberculosis complex, can also be attenuated by the inventive method. Additionally, since vaccination of humans with BCG (an attenuated form of M. bovis) provides some protection against M. leprae, the causative agent of leprosy (see McMurray, D., Mycobacteria and Nocardia, in Baron, supra, at 452: xe2x80x9cvaccination with M. bovis BCG has been effective in some endemic areasxe2x80x9d), in addition to whatever protection it confers against tuberculosis, it is expected that vaccination with attenuated pathogenic mycobacteria such as M. tuberculosis and M. bovis will confer at least some protection against diseases caused by other pathogenic mycobacteria as well.
Nor are vaccines the only use of the attenuated strains of the invention. The physiology of slow-growing pathogenic species of mycobacteria is unique in many respects related to their preferred niche of growth within mammalian macrophages. Because of the risk of infection of laboratory workers, however, many laboratories engaged in mycobacterial research employ instead as models fast-growing species of mycobacteria, which are saprophytic and non-pathogenic. These species are extremely limited in their ability to predict drug efficacy against pathogenic forms, or the efficacy of potential vaccines or diagnostic products for pathogenic species. For example, high-throughput drug screening programs currently use a non-pathogenic mycobacterium, M. aurum, as a model organism to screen potential drug candidates. The attenuated forms of pathogenic mycobacteria provided by the invention are much closer models of the target organisms, and accordingly provide a significantly improved tool for screening for novel antimycobacterial agents.
In addition, subcellular fractions of pathogenic mycobacteria are common components of commercial products, such as the composition referred to as PPD, described above. This mixture of Mycobacterium sp. cellular debris is used on a global scale by health workers for detection of exposure to M. tuberculosis and M. bovis. Such fractions are also commonly used for commercial adjuvants, such as Freund""s complete adjuvant and RibiImmunochem Research""s (Hamilton, Mont.) MPL adjuvant. In addition, these attenuated strains can be used as diagnostics, including use as reagents for the development of monoclonal antibodies to recognize wild type mycobacteria in patient samples. Because of the reduced risk of infection of personnel, the attenuated forms of mycobacteria provided by the invention are a significant improvement over wild type strains for such purposes.
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.